Immuno-modulated replication-efficient vaccinia virus strain

ABSTRACT

The invention refers to new immuno-modulated replication-efficient Vaccinia virus strain (IOVA) and its derivatives for the use in medicine.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created May 28, 2020, isnamed 50322-004001_Sequence_Listing_05.28.20 ST25 and is 9,277 bytes insize.

FIELD OF THE INVENTION

The invention refers to new immuno-modulated replication-efficientVaccinia virus strain (IOVA) and its derivatives for the use inmedicine.

BACKGROUND OF THE INVENTION

Recently, for the first time a virus has obtained market approval to beused as an oncolytic virus in the treatment of human cancer. Such asuccess refocused the scientific interest on viruses in general and alsoon the use of Vaccinia virus for the treatment of cancer. The use ofVaccinia viruses proved already to be effective not only in vaccinationapproaches, e.g. protecting against poxvirus infections in man, but havealso been applied in many other medical treatments and clinical trialsfor e.g. tumour vaccination.

The scientific community knows, from data obtained in the first clinicalstudies with oncolytic viruses that these viruses or designed viralvectors are capable to selectively replicate in cancer cells andactively promote the lysis of such infected cancer cells. However, thedirect lysis of such infected cancer cells is rarely sufficient toeradicate bulky tumours and rarely cures metastatic disease. Theseresults illustrate the increasing need not only for more potent andselective viruses, but also for alternative mechanisms of tumourdestruction in order to avoid recurrence of the disease.

More recently, viruses and viral vectors based on Vaccinia virus weretested as oncolytics and seem to be most promising due to their capacityto destroy tumours through mechanisms different from the direct lysis ofthe tumour cell (Kirn et al., 2009). On the one hand it was demonstratedthat the replication of the virus in tumour-associated endothelial cellsleads to disruption of tumour blood flow, hypoxia, and massive tumournecrosis (Breitbach et al. 2013); on the other hand the replication ofVaccinia virus within the tumour is capable of inducing an anti-tumourimmune response thanks to the release of tumour-associated antigens(TAAs) after lysis, and to an overcoming of the local immunosuppressionexisting within the tumour (Thorne et al., 2010).

These alternative mechanisms proved to be key in clinical evaluations,and highlighted the critical role that the immune system plays indetermining the activity of oncolytic vectors. In randomized clinicaltrials, the most effective viruses proved to be those that express animmuno-activating cytokine in order to optimize the activation of thecellular immune response (Breitbach et al., 2011; Heo et al., 2013).However, even with these more potent cytokine-expressing viruses, somepatients do not elicit an effective immune response against the tumour.Generation of effective anti-tumour immune responses require activationand collaboration of even more and different lineages of immune cells,and the expression of a single cytokine can hardly change the overallimmunity elicited by a virus. Thus, research on the generation of novelstrains with a higher capacity to activate a robust immune response isstill needed.

It is therefore an objective of the present invention to provide newlyadapted and/or more potent viruses and/or viral vectors useful for thetreatment of human diseases, wherein the viruses or vectors uponinfection elicit and/or activate additional and more powerful, but—forsafety reasons—highly specific immune responses in the patient needingsuch treatment.

This object has been solved by the newly identified immuno-modulatedreplication-efficient Vaccinia virus strain as specified in claim 1 ofthe present application. Further embodiments refer to variations of thisnewly identified and/or adapted immuno-modulated Vaccinia virus as wellas vectors, preferred uses and compositions comprising the newlyidentified and/or adapted Vaccinia virus strains are described in thedependent claims.

While studying the immune response to the known Vaccinia virus strains,and particularly in comparison to a well-characterised Vaccinia virusstrain (Western Reserve (WR)) the inventors were able to isolate,generate and characterise a new Vaccinia virus strain. This novel strainshows a clearly improved immunological profile. Without being bound bythe theory, we believe that the novel strain also shows a strongpromotion of an increased immunogenic cell death upon infection oftumour cells and thereby redirects the immune reaction towards theinfected and the not-yet-infected tumour cells.

The strain Vaccinia virus WR is a well-defined virus strain that hasbeen tested as oncolytic due to its high replication capacity in tumourcells and was used for the delivery of GM-CSF for increasing the immuneresponse against tumours. In order to provide vectors with tumourselectivity, different Vaccinia virus genes involved in activating themetabolism of infected cells were successfully deleted, restricting thereplication of the resulting virus vectors to cells with ahigh-replication index. Deletions of genes such as thymidine kinase orviral growth factor have already been included in viral candidatestested in clinical trials (Zeh et al., 2015).

WO2015027163 is one example wherein an oncolytic Vaccinia virus isdescribed, which comprises genomic deletions in some of the typicalviral immune evasion genes. In this particular case the oncolytic viruscomprises deletions in e.g. the B8R, B18R and A35R genes.

EP2136633 is another example, which describes the therapeutic use of aGM-CSF-expressing Vaccinia virus in oncolytic treatment approaches.

In these oncological approaches, Vaccinia virus WR strain is consideredthe current gold standard for the development and for comparison ofnewly developed oncolytic Vaccinia viruses.

However, beside this alleged success there are still some concernsconsidering Vaccinia virus WR as the basis for a therapeutic approach inseverely sick cancer patients. As Vaccinia virus WR was selected in vivoafter multiple passages in mouse brains, which factually resulted in avirus with high replicative capacity in mouse cells, but also in analarming virulence due to neurotropism and neurotoxicity, which mayhinder its safe use in the treatment of cancer patients.

Nevertheless, in the context of this application Vaccinia virus WR waschosen as comparison to the newly developed Vaccinia virus strain of thepresent invention, wherein the main goal of the present invention is toprovide a novel immuno-modulated replication-competent Vaccinia virusstrain with a substantially improved immunological profile and highlytic and/or oncolytic capability.

An additional comparison with the highly attenuated MVA (ModifiedVaccinia Ankara) strain of Vaccinia virus seems not to be useful, as MVAis characterised by its host rang restriction or in other words itsdefective replication in cells from mammalian hosts. MVA has beenbroadly used for delivering antigens for vaccination against pathogens(for review see Volz & Sutter, 2017) and has been characterisedregarding its biological and immunological profile (Meyer et al., 1991).Additionally, MVA is a highly modified Vaccinia virus unable toreplicate in mammalian cells and has been previously used forvaccination against tumours by delivering tumour-associated antigens(TAA) (Zhang et al., 2012). Regardless of the fact that this virusseemed to show an ideal safety profile and was demonstrated to beeffective generating an immune response against the expressed tumourantigens in vivo, most patients did not show any verifiable anti-tumourresponse; it seems that the elicited immunity is hindered by the immunesuppression that occurs locally within the tumour, leading to anineffective anti-tumour response (Marigo et al., 2008).

Thus, although progress could have been stated towards an improvedimmunotherapy, there is still need for more effective Vaccinia virusstrains to be used in virotherapy of cancer in order to establish aneffective immune response capable of eradicating cancers.

The present invention describes now a further step towards this goal anddiscloses a novel Vaccinia virus strain, which is replication competentin human and mouse cells and is—according to the best knowledge of theinventors—even the first Vaccinia virus in general that causes animmunologically relevant calreticulin (CRT) translocation to the outermembrane and thus causes an exposure of calreticulin on the outer cellmembrane of infected cells, thereby initiating a potent and an improvedimmune response.

Calreticulin (CRT) seems to be a multifunctional protein and has beendescribed to be involved in transcription regulation ofhormone-responsive DNA-elements and in the maturation of MHC class Iproteins; it has also been described to bind and inactivate Ca²⁺ ions ormisfolded proteins in the endoplasmic reticulum, and primarily to belocated in the storage compartments of the endoplasmic reticulum (ER).CRT represents the most abundant protein of the ER lumen, but a fractionof the protein can translocate from the ER lumen to the surface of thecell in case of cell death; more specifically, it has been suggestedthat CRT acts as an “eat-me” signal on the cell surface (Gardai et al.,2005), mediating the engulfment by CD91-positive cells (mostlymacrophages and dendritic cells (DCs)).

The novel Vaccinia virus strain is described to be replication competentin mammalian cells. It is believed—without being bound by thistheory—that this characteristic depends inter alia on the presence andfunctional activity of the K1L gene in the viral backbone. Inparticular, it has been shown that the novel strain replicates well inmammalian and particularly in human and mouse cell lines, for example incancer cells such as HeLa cells, 143B cells, CT26 cells, LLC1 cells orMCF-7 cells. In comparison to these results it should be noted that MVAis known not to replicate in these cells or in other cells of mammalianorigin.

Furthermore, the novel Vaccinia virus strain shares severalhouse-keeping genes with all and/or at least some of the other Vacciniavirus strains. It is, however, quite distinct in its immunologicalprofile and is named IOVA (Immune-Oncolytic Vaccinia) in the context ofthe present application.

Many genes of the Vaccinia virus genome have been described to be immuneevasion genes (Smith et al., 2013), which form and determine theimmunological profile of the different virus strains. Over the years,studies have been conducted to identify the potential mechanism ofaction of such immune evasion genes. According to the results obtainedthese immune evasion genes express—inter alia—soluble receptors forcytokines, which compete with the natural receptors and, reduce theefficacy of a cytokine-induced immune response. Other genes block theintracellular pathways for activating different immune-activating genesand, thus, also interfere and reduce a virus specific immune response.

For a short summary, Table 1 lists the presently identified immuneevasion genes of Vaccinia virus and their presence or absence indifferent Vaccinia strains. It also indicates a potential function ormechanism of action of the corresponding gene product.

TABLE 1 Known Vaccinia immune modulators compared to IOVA sequence ORFGENE PRODUCT IOVA CVA MVA COP WR C12L Interleukin-18-binding protein + +− + + C10L Binds DNA--PK − − − + + C9L Ant-like protein − − − + + C8L*Interleukin-18 binding protien + + + − + C6L IRF3 binding + + + + + C5LKelch-like protein + + − + + C4L NF-κB inhibitor − − − + + C3LComplement control protein + + − + + C2L Kelch-like protein, modulator −− − + + of inflammation N1L Inhibitor of TNF-R and TLR − + − + +signaling N2L IRF3 inhibitor − + − + + M1L Apoptosis inhibitor − − − + +K1L NF-κB inhibitor + + − + + K2L Serine protease inhibitor;SPI-3 + + + + + K3L eIF2α-like protein + + + + + K7R NF-κB/IRF3inhibitor + + + + + F1L Anti-apoptotic, inflammasome + + + + + inhibitorF3L Kelch-like protein + + − + + E3L Double-strandedRNA-binding + + + + + protein H1L Dephosphorylates STAT1 and + + + + +STAT2 (VH1) D9/D10 Cleavage of 5-methylated caps + + + + + on mRNAs A35RInhibitor of MHC class II + + + + + A38L CD47-like protein + + + + +A40R C-type lectin-like protein + + + + + A41L Secretedchemokine-binding + + + + + protein A42R Profilin-1-like + + − + + A44L3 β-hydroxysteroid + + + + + dehydrogenase A46R NF-κB/IRF3inhibitor + + + − + A49R NF-κB inhibitor + + + + + A52RToll/IL1-receptor inhibitor − + − + + targeting IRAK2/TRAF6 A53R SolubleTNF receptor (CrmC) − + − − − A55R Intracellular kelch protein − − − + +immunomodulator A56R Haemagglutinin/Blocker of − + + + + NK cell lysisB7R Chemokine-binding domain + + − + + protein SCP-3 B8R SecretedInterferon-γ binding + + − + + protein B13R/ Inhibitor of caspases − − −− + B14R (SPI-2, CrmA) B15R Secreted IL-1β binding protein + + − + +B16R Interleukin-1 beta receptor + + + − + B19R Soluble and cellsurface + + − + + interferon-α/β receptor B21R* Chemokine-binding domain− − − − + protein SCP-1 ORF: Nomenclature of Vaccinia COP strain.*Nomenclature in CPXV-GRI. +: Full and functional; −: Completely orpartially deleted (non-functional).

The nomenclature of the open reading frames (ORF) used in Table 1 refersto the naming system established for the Vaccinia Virus Copenhagen (COP)strain (Goebel et al. 1990) and is identified by letters of the alphabetassigned to DNA fragments of the viral genome generated by therestriction endonuclease HindIII descending in size. Exceptions are theORF C8L and B21R with letters referring to DNA fragments in the cowpoxvirus CPXV-GRI genome, due to their absence in the Vaccinia Virus COPstrain. The different ORF in such HindIII fragments are identified bynumbers. The letter at the end indicates the direction of transcription:R for right, L for left.

In addition, Table 1 provides a comparison regarding functionality ofthe so far identified immune evasion genes with the well-characterisedVaccinia virus strains COP, WR, MVA, as well as the MVA ancestor strainCVA. It is indicated in Table 1, whether the different genes arefunctional (indicated by “+”), partially/fully deleted and functionallyinactive (indicated by “−”) in the different virus genomes.

The novel Vaccinia virus strain, as described herein and in thefollowing identified as IOVA, has an immunological profile of its own.It shares some of the functionally expressed genes with CVA or WR andalso shares some of the functionally inactive genes with MVA. This hasbeen confirmed by the inventors also on a sequence level, comparing thefull-length sequence of one isolate of the novel Vaccinia virus IOVAwith the published sequences of WR, COP, CVA, and MVA.

The inventors also developed a PCR assay for easy identification of thenovel IOVA strain. For this, a fragment of the DNA of any Vaccinia virusto be tested can be amplified with PCR oligonucleotides (primers)specifically binding in the region or sequence of the C2L ORF (OpenReading Frame) on the one hand and in the region or sequence of the N2LORF. A PCR performed by standard parameters will produce for mostVaccinia viruses an amplification fragment comprising the sequence ofthe ORF C2L-C1L-N1L-N2L. These PCR products are double-stranded DNAfragments and as such are then subject to an enzymatic treatment withthe restriction endonuclease BstXI. As a result, the specific DNAproducts obtained in e.g. Vaccinia strains WR, COP and CVA would not becleaved and present a single band of around 3280 bp (molecular weight)upon agarose gel electrophoresis. In contrast, the specific IOVA DNAproduct will be cleaved by BstXI and will present two bands in theagarose gel with molecular weights of about 2360 and 920 bp. Theexplanation is that the novel IOVA virus carries a highly specificmutation in the N1L ORF leading to a functional inactive N1L and a newBstXI restriction site. In the case of MVA, the C2L gene sequence isdeleted in the MVA genome, accordingly there will be no amplification ofa DNA product by this specific PCR.

As can be seen in Table 1 and in the data presented in this application,IOVA—itself as well as all derivatives thereof—although it shares manygenes and/or functional deletions with CVA, WR and MVA, is uniqueregarding not only its safety features but also its immunologicalprofile.

This immunological profile—being the essential core of this invention—iscorrelated inter alia with the presentation of calreticulin on the outermembrane of a virus-infected cell.

Thus, the immunological profile of the novel strain is described toelicit—inter alia—a measurable calreticulin presentation on the outermembrane of a virus-infected cell. It is believed—without being bound bythis theory—that this characteristic depends on functional inactivity,partial or full deletion of at least one of the immune evasion genesselected from the group consisting of the B21R*, C10L, C9L, C4L, C2L,N1L, N2L, M1L, A26R, A51R, A52R, A55R, A56R, and B13R/B14R (Vacciniavirus Cop strain nomenclature except*, which correspond to CPXV-GRInomenclature) in the viral backbone.

As a consequence of these functionally inactive, partially or fullydeleted genes, the newly generated virus has not only lost some of itscapability to escape the immune response of the host, but additionally ashift in immune-evasion strategies has been introduced.

TABLE 2 Distinction between IOVA and WR ORF GENE PRODUCT IOVA WR C10LBinds DNA-PK − + C9L Ant-like protein − + C4L NF-κB inhibitor − + C2LKelch-like protein, − + modulator of inflammation N1L Inhibitor of TNF-R− + and TLR signaling N2L IRF3 inhibitor − + M1L Apoptosis inhibitor − +A52R Toll/IL1-receptor − + inhibitor targeting IRAK2/TRAF6 A55RIntracellular kelch − + protein immunomodulator A56RHaemagglutinin/Blocker − + of NK cell lysis B13R/B14R Inhibitor ofcaspases − + (SPI-2, CrmA) B21R* Chemokine-binding − + domain proteinSCP-1 ORF: Nomenclature according to Vaccinia virus - COP strain. +:Complete and functional; −: Completely or partially deleted orfunctional inactivated.

Particularly interesting in this context is the effect that thecalreticulin protein, which normally is strictly internal and located inthe endoplasmic reticulum, but can—when translocated to the cell surfaceof cancer cells after the infection with the novel Vaccinia virusstrain—initiate and intensify a specific immune reaction towards thevirus-infected cells. Calreticulin is a Danger Associated MolecularPattern (DAMPs) described to act as an “eat-me” signal when presented onthe outer membrane and can been used as a marker of an immunogenic celldeath. In comparison to Vaccinia virus WR the newly generated Vacciniavirus strain, IOVA, lyses or kills cancer cells at a much lower doseand, thus, much more efficient (FIG. 3A).

It was shown by the inventors that in both human and mouse cell lines,WR was only able to kill 70-80% of cultured tumour cells; around 20-30%of the cells escaped the virus-mediated destruction and remainedmetabolically active even with highly increased multiplicity ofinfection.

On the contrary, a derivative of the novel IOVA virus destroyed at leastand even more than 95% of the cells in all cell lines tested; veryastonishing, in the human cell line HeLa, the novel IOVA derivativedemonstrated a highly improved cytotoxicity, being at least 40 timesmore efficient than Vaccinia virus WR, although with regard to growthcapacity of IOVA in HeLa it was very similar to that of Vaccinia virusWR. This more complete efficiency to kill virus-infected cells reducesparticularly the risk of tumour remission and, thus, is highlyadvantageous.

Accordingly, it is not only highly advantageous to use the novelVaccinia virus strain for the infection of cell cycle activated cellsand/or tumour cells, and in this context particularly for the use asoncolytic virus or for use in the treatment of cancer, but also forvaccination purposes and more general immune stimulation purposes, thusfor a use in medicine in general.

Additionally, according to a further embodiment, the novel Vacciniavirus strain IOVA comprises functionally inactive A56R gene. The lack ofexpression of the product of this gene correlates with the capacity ofthe virus to fuse infected cells with neighbouring cells and, i.e. toform syncytia upon infection of mammalian cells and tumour cells.

Syncytia formation has been described in many viral infections and, inmost of these infections, its formation is as consequence of themembrane destabilization caused by a cytopathic effect; however, someother viruses encode proteins that promote this syncytia formation as amechanism to spread the infection without being a target to neutralisingantibodies. Interestingly, the A56R gene product is expressed byVaccinia virus WR in order to inhibit such syncytia formation (FIG. 1 ).

The finding that the novel Vaccinia virus strain IOVA causes syncytiaformation in cancer cells is considered as a potentially interestingphenotype for oncolytic viruses as syncytial cell death has beendescribed to be highly immunogenic: fusion of tumour cells triggers therelease of exosome-like vesicles associated with an effective release oftumour antigens, which can efficiently be uptaken by dendritic cells andthus improve a highly specific immune reaction.

Additionally, according to a further embodiment, the novel Vacciniavirus strain IOVA comprises functionally inactive A26R and A56R genes.The lack of expression of the product of these genes correlates with aneven improved formation of syncytia upon infection of mammalian cellsand tumour cells and thus, also correlates with the capacity of thevirus to fuse infected cells with neighbouring cells.

To investigate syncytia formation as a possible mediator of IOVAvirus-specific anti-tumour efficacy and the effect of the deletion ofA56R alone or in combination with the deletion of A26R in such fusion, asingle A56+ IOVA virus variant was engineered by replacing the mutatedA56R gene and creating the IOVA/A56+ (FIGS. 1 and 6 ). When the A56Rgene expression is restored, the IOVA/A56+ virus conserves a certaincapacity to fuse tumour cells, although the number of fused cells isreduced compared to the IOVA/A26−/A56− virus (FIG. 6 ); this indicatesthat both genes may interact to fully inhibit the fusion of infectedcells.

Syncytia formation is known to have a negative impact on the productivereplication of other candidate oncolytic viruses, e.g. derived frommeasles virus or adenovirus. Surprisingly, syncytia formation in thecontext of IOVA viruses has been demonstrated to have a minimal impacton the replicative capacity, and very similar viral yields were obtainedfrom cells infected with IOVA/A56− and IOVA/A56+ (FIG. 2 ).

Surprisingly, the formation of syncytia had a remarkable positive impacton cancer cell cytotoxicity according to the present invention: in allcell lines tested the syncytia-forming virus performed a more effectivekilling of cancer cells, especially in 143B and MCF-7 cancer cell lines,where IOVA/A56+ behaved similarly to Vaccinia virus WR with reducedkilling of the cultured cancer cells (FIG. 3 ) Thus, syncytia formationmediated by IOVA/A56− and also IOVA/A56−/A26− can contribute to enhanceddestruction of cancer cells.

A large-plaque phenotype was observed after the infection of cancercells. Both IOVA/A56− and IOVA/A56+ viruses present an increasedcapacity to kill and to spread in cancer cells as shown by increaseddiameter of the plaques generated after the infection of a single cancercell (FIG. 7 ).

In terms of inducing immunogenic cell death, syncytia formation is alsoindependent from the induction of CRT translocation, and also from thelater described additional secretion of further DAMPs such as HMGB1 andATP, which was very similar for both A56− and A56+ viruses (FIG. 4 a-c).

Interestingly, in the context of the present invention, it was shownthat the phenotype of syncytia formation, which on its own already hasan enhancing cytopathic effect in cancer cells, in addition to theindependent exposure of calreticulin on the outer membrane of theinfected cell, increased even more the antitumor effectiveness of theimmune-modulated Vaccina virus strain IOVA as herein described.

Accordingly, the novel Vaccinia virus strain may contain additionallyfunctionally inactive A56R and/or A26R genes and thus, cause syncytiaformation and induce the fusion of tumour cells. In combination with theenhanced presentation of calreticulin on the membrane of the infectedcell this leads to an even greater lytic and, thus, anti-tumour activityand results in consequence in enhanced destruction of cancer cells,tumour masses and eventually the generation of stronger immune responsesagainst tumours.

In order to increase the safety of the novel IOVA virus, derivativesthereof and/or viral vectors thereof, additional modifications have beenor may be introduced to restrict the replication to a specific cellrange only.

Therefore, according to another embodiment the novel Vaccinia virusstrain, IOVA, is modified to have a host range or, in other words,cell-range restricted replication competence in replicating cells and/orcancer cell.

In this context the term “replicating cell” comprises cells that have anactivated cell-cycle and replicates at a high index. The cells fallingunder this definition comprise cells that are continuously replicatingsuch as cancer cells, but also cells belonging to e.g. the activatedimmune system and optionally having the potential to infiltrate tumours.

Further the term “cancer cell” as used hereinafter comprises cells thatdivide inexorably and uncontrolled and have the capability to supersedeor dispossess normal tissue by e.g. forming solid tumour mass or byflooding the blood and/or other body cavities with abnormal cells.Furthermore, the term “cancer cell” optionally refers also to cells thatexpress and can be identified by their expression of known tumour markergene products.

To improve the cell range specificity of the novel Vaccinia virusstrain, it was shown that by inactivating one or more of the so-calledhouse-keeping genes selected from the group consisting of F4L, J2R andC11R, a cell-range restricted replication competence can be obtained.For example, a virus with an inactivated or mutated J2R gene will notexpress the viral thymidine kinase and, thus, its effective replicationwill be limited to cells with a continuously activated cell-cycle oralternatively, to tumour cells. According to the present invention, suchinactivation has been exemplarily performed by the insertion of analternative expression cassette (expressing a tracer colour) onto thelocation of the TK gene, which in Vaccinia nomenclature is referred toas J2R.

Similarly, also the functional inactivation, deletion or mutation of theC11R, the F4L alone or in combination with each other or J2R, will limitthe replication competence of the Vaccinia virus strain, according tothe present invention, to cell-cycle activated and/or tumour cells.Thus, such addition of mutations or deletions to the viral genome inorder to functionally inactivate one or more gene of the groupconsisting of F4L, J2R and C11R having the above-described consequenceof a host range restriction, clearly improves the safety features forthe novel virus strain.

Therefore, the cell range restricted Vaccinia virus strain, IOVA, isparticularly advantageous for use in medicine because of its improvedsafety features.

It has been further shown by the inventors that the novel Vaccinia virusstrain of the present invention is—compared to Vaccinia virusWR—surprisingly capable of inducing an immunogenic cell death uponinfection.

In this context the term “immunogenic cell death” is to be understood asa form of cell death that is able to initiate and activate an immuneresponse against the dying cell and is characterized by the release orexposure of damage-associated molecular pattern (DAMP) molecules, whichcan arouse an immune response against neo-antigens, both microbial andoncogenic (Galluzzi et al., 2017). Among a long list of DAMPs releasedor exposed by dying cells that may mediate an immune response to tumourcells, the exposure of calreticulin on the cell surface of cells, andindependently the release of high-mobility group box 1 protein (HMGB1)or ATP into the extracellular space have been described.

Immunogenic cell death can further be described as a form of cell deaththat expose or release different DAMPs in order to activate dendriticcells (DCs) and consequent activation of specific T cells againstantigens present in the dying cell. It is not triggered from theoutside, as e.g. apoptosis, which is for example defined asnon-immunogenic and even tolerogenic cell death and cannot be defined bythe same DAMPs.

With the background knowledge of such markers, it was possible for theinventors to actually describe and classify the type of cell deathcaused by the viruses. And consequently, it was clearly demonstratedthat in comparison to Vaccinia virus WR, the novel Vaccinia virusstrain, IOVA, did cause not only an increased lytic and cell killingeffect upon infection, but it was also possible to show by measuringsome of the DAMPs, namely HMGB1 and ATP, and by measuring the amount ofCRT presented on the outer cell membrane that the infected cells dieddue to an immunogenic cell death.

This proves impressively that the novel Vaccinia virus strain, whichreplicates in mammalian cells and specifically in tumour cells orcell-cycle activated cells, is highly useful not only as vaccine oradjuvant for vaccination purposes, but also in the context of furtherimmunotherapeutic or immuno-oncological treatments, particularly asoncolytic virus for its use in the treatment of cancer.

In summary, the novel Vaccinia virus strain IOVA as well as itsderivatives, vectors and recombinants have the ability to replicate inmammalian cancer lines and can be described as being lytic or, in caseof cancer cells, oncolytic, as they elicit—compared with the goldstandard Vaccinia virus WR—an increased capacity to destroy cell-cycleactivated cells and/or tumour cells.

Importantly, IOVAs and derivatives or recombinants thereof areparticularly suitable to induce an immunogenic cell death upon infectionof cells and thereby are highly promising to be used inimmunotherapeutic approaches. They are particularly suitable as novelplatform viruses for a safe and efficient immunotherapeutic orimmuno-oncologic treatment.

According to a further embodiment, the present application also providesan isolated IOVA virus characterised by its nucleic acid sequence. Thesequence information of the isolated IOVA may comprise base pairmodifications, which—however—do not affect the immunological profile ofIOVA. Thus, an IOVA virus can be identified by sequence analysis and thepresence of at least one or more functionally inactivate or deleted ORFselected from the group consisting of the J2R, C11R, F4L, B21R*, C10L,C9L, C4L, C2L, N1L, N2L, M1L, A26R, A51R, A52R, A55R, A56R, andB13R/B14R.

According to one further embodiment IOVA and its derivatives can beidentified by the presence of a unique nucleotide sequence stretch ofthe C2L-C1L-N1L-N2L (SEQ ID NO.: 1), which introduces in the N1L ORF anewly generated BstXI restriction enzyme site. In this sequence, C2Lincorporates a deletion of 51 nucleotides distributed in 3microdeletions; N1L incorporates a 2 nucleotides deletion thatincorporates an early termination codon; and N2L incorporates a 15nucleotides deletion that codifies for a shorter N2L version. Thus, thesequences shown in SEQ ID No.1 is a unique sequence of IOVA andrepresents the unique stretch of for the C2L-C1L-N1L-N2L region of IOVA,incorporating microdeletions in some of the ORFs, but not completedeletions of such genes and no microdeletion of the C1L ORF, which isfunctional.

Additionally, the isolated IOVA—according to one embodiment—isconsidered to be a platform technology, which allows the generation ofrelated derivatives or recombinants, identified by the PCR analysis asmentioned above or a direct sequence comparison of at least one or moreof the viral housekeeping or the functional inactive immune evasiongenes selected from the group of ORFs consisting of the K1L, A56R, A26R,J2R, C11R, F4L, B21R*, C10L, C9L, C4L, C2L, N1L, N2L, M1L, A51R, A52R,A55R and B13R/B14R.

According to a further embodiment the present application also providesa viral vector derived from IOVA. This viral vector comprises the sameset of functionally active and functionally inactive genes as IOVA. Theterm “viral vector” in the context of this application also includes twoor more vector molecules each comprising or carrying parts or ranges ofthe set of functionally active and functionally inactive genes of IOVAworking in concert to transfect a cell enabling IOVA to be produced fromsuch cell.

IOVA itself, the isolated virus, the nucleic acid sequence of IOVA and aviral vector(s) comprising the IOVA specific nucleic acid sequence areconsidered a platform technology.

Said IOVA platform comprises also derivatives of IOVA, which can beidentified by PCR and a restriction enzyme digest with the BstXI enzymeand which still employ the same functional features and characteristicsas IOVA, particularly the replication competence in mammaliancell-cycle-activated cells, the calreticulin translocation to the outermembrane and/or independently the induction of syncytia formation ininfected cells.

Said IOVA platform further comprises recombinants of IOVA. The backboneof IOVA contains several well-described Vaccinia virus insertion sitesfor transgenic insertions into its genome. Due to its close relation toVaccinia virus the skilled practitioner knows and is capable of usingone or several locations for the insertion of transgenes, such as e.g.genes encoding tumour antigens, tumour associated antigens, diseaseassociated antigens and/or pathogen derived antigens.

According to a further embodiment, the invention thus provides arecombinant or transgenic IOVA or a derivative or viral vector thereof.As in every platform technology, the introduction of additional genes orgenetic information in well-described insertion sites does not affectthe main characteristics of the claimed platform members, theseindependently being (i) the induction of a fusion or syncytia formationof infected cells, (ii) the induction of a prominent release or exposureof DAMPs, particularly of calreticulin and/or (iii) the induction of animmunogenic cell death of the infected cell, which may contribute to aneven greater immunological effect or in case of the infection of tumourcells a more effective anti-tumour activity through enhanced destructionof tumour cells.

It is believed—without being bound by the theory—that the more effectiveanti-tumour activity is particularly due to a stronger immune responseagainst such infected cells, which may be triggered by presentation ofcalreticulin and/or an increased release of other DAMPs, such as HMGB1or ATP. Released HMGB1 can bind to TLR4 and RAGE and triggerproinflammatory responses, while released ATP seems to act also as a“find-me” signal for immune cells.

In summary, the novel Vaccinia virus strain IOVA as well as itsderivatives, vectors and recombinants have the ability to replicate inmammalian cancer lines and can be described as being lytic or, in caseof cancer cells, oncolytic, as they elicit—compared with the goldstandard Vaccinia virus WR—an increased capacity to destroy cell-cycleactivated cells and/or tumour cells.

IOVA or its derivatives, due to its unique immunological profile and itssafety features, which are comparable or even better than in alreadywell-established Vaccinia virus strains, can thus be used in medicine,particularly in oncological approaches or as an oncolytic or as avaccine against cancer or other pathogens.

Importantly, IOVA viruses and derivatives or recombinants thereof areparticularly suitable to induce an immunogenic cell death upon infectionof cells and are thereby highly recommended to be used inimmunotherapeutic approaches. They are particularly suitable as novelvirus platform for a safe and efficient immunotherapeutic orimmuno-oncologic treatment.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1 . Syncytia formation in cancer cells after infection with IOVAviruses. Fluorescent photomicrographs (40×) are shown after infection of(a) human or (b) mouse cancer cell lines (24 hours post-infection). HeLaand CT26 cells were infected with an MOI of 0.5. 143B, MCF-7, and LLC1were infected with an MOI of 5. mCherry is expressed from all theviruses under the P11 promoter. Massive fusion of cells (syncytium) canbe observed when cells were infected with IOVA/A56−.

FIG. 2 . Viral production of IOVA viruses in human and mouse tumourcells. (a) Human and (b) mouse tumour cell lines were infected withWR/TK−, IOVA/A56−, or IOVA/A56+ at an MOI of 5, and progeny was measuredby plaque-assay at different time points. Viral yield was evaluated inquadruplicate for each cell line, by carrying two independentexperiments. Means+SD are plotted. *, significant p<0.05 compared withWR/TK−. #, significant p<0.05 compared with IOVA/A56+.

FIG. 3 . IOVA viruses present increased cytotoxicity to tumour cells.Cancer cells were infected with WR/TK−, IOVA/A56−, or IOVA/A56+ at dosesranging from 100 to 0.0005 PFU/cell. At day 3 after infection, viabilityof cells was determined. Both human (a) and mouse (b) cancer cells weretested. Four different replicates were quantified for each cell line andmean±SD of each MOI is depicted.

FIG. 4 . Induction of Immunogenic Cell Death by IOVA viruses. (a)Analysis of Calreticulin expression on the surface of infected cells.Indicated tumour cell lines were infected with WR/TK−, IOVA/A56−, orIOVA/A56+ with an MOI of 5, and 24 hours after infection Calreticulin+cell populations were determined by flow cytometry. Uninfected cells(Mock) and Staurosporin 1 μM were used as negative and positivecontrols, respectively. (a) Percentage of Calreticulin+ cells. Values ofindividual replicates and means±SEM of the different treatments areplotted. (b-c) Concentration of HMGB1 (b) and ATP (c) in cellsupernatant after infection with IOVA viruses. ELISA assays and ENLITENATP assay system were utilized, respectively, to determine suchconcentrations at 24 hours after infection (MOI of 5) of indicatedtumour cell lines. Data were obtained in quadruplicate and are plottedas fold change versus WR/TK−+SD. *, significant p<0.05 compared withWR/TK−. #, significant p<0.05 compared with Mock.

FIG. 5 . PCR Assay for Virus identification. FIG. 5 a : Sequence of thePCR product employed in the PCR Assay. FIG. 5 b : Results of the PCRassay clearly identify the sequence of IOVA strain as the assaygenerates a unique pattern of two bands (2320 and 920 bp) for IOVA, incontrast to all the other Vaccinia virus strains, which present a singleband of 3300 bp for WR, COP and CVA. MVA strain generates no amplifiedproduct due to a C2L deletion.

FIG. 6 . Number of nuclei in syncytia after infection with IOVA viruses.Human tumour cell lines were infected with WR/TK−, IOVA/A56−/A26−, orIOVA/A56+/A26− at an MOI of 5. At 16 hours post-infection, cultures weredyed with Hoechst 33342 and the number of nuclei in one syncytium werecounted under the microscope. Values of individual replicates andmeans±SEM are plotted. ***, significant p<0.0001 compared with WR/TK−.

FIG. 7 . Size of the plaques in cancer cells. Cancer cell monolayerswere infected with indicated viruses for 1 hour at an MOI of 0.0001 andcultivated for 4 days covered with a 1:1 mixture of culture media and 1%carboxymethylcellulose. After fixation and staining with crystal violet,the diameter of the plaques was determined. Values of individualreplicates and means±SEM are plotted. *, significant p<0.05 comparedwith WR/TK−. #, significant p<0.05 compared with IOVA/A56−.

EXAMPLES Example 1: Deletions in IOVA Genome

A novel Immune-Oncolytic Vaccinia virus (IOVA) strain is generatedincorporating a deletion in the thymidine kinase (TK, J2R) gene in orderto confer selective replication in cancer cells. Additionally, themCherry gene has been cloned into the TK site under the control of theVaccinia virus-specific promoter P11 in order to monitor virusreplication.

In addition, the newly generated IOVA contains several deletions orfunctional inactivations among genes considered to be immune modulatorsand selected from the ORF of B21R*, C10L, C9L, C4L, C2L, N1L, N2L, M1L,A26R, A51R, A52R, A55R, A56R, and B13R/B14R. The functions of theproteins coded for these genes are summarized in Table 1 above.

The IOVA genome is further characterised by the inclusion of a mutatedversion of the A26R and/or the A56R gene. The presence of A26 protein inthe virion prevents direct virus-cell fusion mechanism and its deletionhas been associated with induction of syncytia. A56R encodes a viralregulatory protein with haemagglutination activity, and its inactivationin Vaccinia virus is believed to result in viruses with a fusogenicphenotype. All gene deletions or partial deletions as well as allfunctional inactivation or gene insertions have been confirmed bysequencing.

Example 2: Syncytia Formation Induced by IOVA

In order to evaluate the pros and cons of syncytia formation for tumourdestruction, the inventors restored by homologous recombination thewild-type Vaccinia virus A56R gene sequence in the novel IOVA genome.The resulting virus was named IOVA/A56+, in comparison to the IOVA withthe truncated A56R version, which was named IOVA/A56−.

Upon infection, it was observed (FIG. 1 ), that cells infected with theIOVA/A56− virus fuse with neighboring cells, and a formation of hugesyncytia could be clearly observed in both human and mouse tumour celllines traced by the mCherry expression. As hypothesized, the expressionof wild-type A56 in IOVA/A56+ restored a phenotype very similar toVaccinia virus WR strain and did not lead to syncytia formation.

Additionally, it was observed that The expression of wild-type A56 inIOVA/A56−/A26− may be described also as only partially blocking theformation of syncytia, as still a fusion of up to 10 cells could beobserved after infection with IOVA/A56+/A26− (FIG. 6 ).

Example 3: Replication Competence of IOVA

We tested the replication competence of IOVA in comparison with thestandard strain Vaccinia virus WR in a wide panel of human and mousecancer cell lines.

For monitoring the replication of the newly generated IOVA, thereplication capacity of two IOVA virus isolates (A56− and A56+) andWR/TK− as a control was tested in several human and mouse cancer celllines.

For this, human and mouse tumour cell lines were infected with WR/TK−,IOVA/A56−, or IOVA/A56+ at an MOI of 5, and progeny was measured byplaque-assay at different time points. Viral yield was evaluated inquadruplicate for each cell line, by carrying two independentexperiments (previously described in: Rojas J J et al., Cell Rep. 2016).

As shown in FIG. 2 , both IOVA viruses (A56− and A56+) performed agrowth curve very similar to control strain WR/TK− in most cell linestested, with only a slight reduction of the yield at early time pointsfor the syncytia-forming IOVA.

Example 4: Cytotoxicity of IOVA

We examined the cytotoxic effect of an IOVA infection in comparison withthe standard strain Vaccinia virus WR in a wide panel of human and mousecancer cell lines.

For this, various cancer cell lines were infected with WR/TK−,IOVA/A56−, or IOVA/A56+ at doses ranging from 100 to 0.0005 PFU/cell. Atday 3 after infection, viability of cells was determined. Both human andmouse cancer cells were tested. Four different replicates werequantified for each cell line and mean±SD of each MOI is depicted.

Interestingly, infections with the IOVA viruses resulted in clearlyenhanced levels of cytotoxicity in cancer cell compared to Vacciniavirus WR (FIG. 3 ). Vaccinia virus WR was able to kill around 70-80% ofthe cancer cells in culture, even when at the highest multiplicity ofinfection (MOI). On the contrary, IOVA/A56− virus was able to killbetween 95-100% of the cultured tumour cells and reduced the EC50(amount of virus necessary to kill 50% of the cells).

Surprisingly, IOVA/A56− decreased the amount of virus required to reduce50% of cell culture viability of HeLa cells by more than 40 foldcompared to Vaccinia virus WR (WR/TK−). IOVA/A56+ also presented aphenotype with enhanced cytotoxicity for cancer cell in vitro; theA56-restored virus killed HeLa, CT-26, and LLC1 cells at similar ratesas IOVA/A56−. Yet in 143B and MCF-7 cells the cytotoxicity was verysimilar to that obtained with WR/TK− infection, suggesting thatvirus-mediated big syncytia formation may contribute to enhanceddestruction of tumours.

Example 5: Large Plaque Phenotype of IOVA Viruses in Cancer Cells

An increased size of the plaques in cancer cells has been associated toa better spread of the virus throughout the tumour and to a higherantitumor activity. For testing the plaque size of IOVA viruses, a panelof cancer cell lines were infected at a MOI of 0.0001 and, after 1 hourof infection, the infected cells were cultured with acarboxymethylcellulose overlay. For 4 after the infection, the cultureswere fixed and dyed with crystal violet and the diameter of the plaqueswere determined.

As shown in FIG. 7 , both IOVA/A56− and IOVA/A56+ induced larger plaquesin all cancer cell lines tested compared to WR/TK− virus control. InHeLa cells, plaques after infection with IOVA viruses were as a mean 40%larger compared to WR/TK−. Impressively, very large plaques could beobserved after infection of 143B and MCF-7 with IOVA viruses, withplaques 2 times the diameter of plaques generated by WR/TK− in the caseof 143B cells and plaques 2.6 time larger in the case of MCF-7.

With regard to plaque size, the generation of big syncytia by IOVA/A56−do not have a significant influence except in the case of MCF-7 breastcancer cell line, were IOVA/A56+ virus generated plaques 1.4 timessmaller compared to IOVA/A56−.

Example 6: IOVA Viruses Induce Presentation of Calreticulin on CellularMembrane and Immunogenic Cell Death of Cancer Cells

In order to test whether the IOVA viruses may be able to elicit andpotentiate an immune response against cancer cells, the inventorsinitially analysed by flow cytometry the exposure of calreticulin (CRT)on the surface of human cancer cells after infection with IOVA/A56−,IOVA/A56+, or the control virus WR/TK−.

For this, cells were infected with an MOI of 5 and, 24 hours after virusinfection, detached using a non-enzymatic cell dissociation solution.Calreticulin was detected by incubating the cells for 1 hour at 4° C.with a human anti-calreticulin-AlexaFluor405 antibody (Abcam, Ref N°ab210431). Uninfected cells and staurosporin (1 μM) were used asnegative and positive controls, respectively.

Upon infection of HeLa cells, WR/TK− induced a surface-exposure of CRTon around 15% of the cells (FIG. 4 a ); on the contrary, surprisinglyhigh levels of more than 80% of the cells expressed CRT on the surfaceupon infection with both IOVA viruses. Similarly, exposure of CRTincreased from around 35% (with WR/TK−) to almost 90% (with IOVAviruses) in 143B cells, and from 3% to more than 72% in MCF-7 cells.

In order to further investigate the possible induction of a pronouncedimmunogenic cell death upon infection with IOVA viruses, the release ofHMGB1 and ATP was determined using an ELISA assay and aluciferase-mediated ATP assay system, respectively.

In all cell lines tested and with both IOVA/A56− and IOVA/A56+,significantly higher concentrations of HMGB1 could be detected in thesupernatant of infected cells compared with cells infected with WR/TK−(FIG. 4 b ), with an increase ranging from 1.23 times (143B cells,IOVA/A56−) to 1.68 times (MCF-7 cells, IOVA/A56+). ATP concentration onthe supernatant of infected cells was also increased when infected withthe IOVA viruses compared to the levels after infection with WR/TK−(FIG. 4 c ), with an increase ranging from 1.12 times (143B cells,IOVA/A56+) to 2.27 times (HeLa, IOVA/A56−).

These results indicated that IOVA virus, but not Vaccinia virus WR,induces an immunogenic cell death of infected human cancer cells. Thus,IOVA can be suggested as a particularly promising candidate virus thatmay represent a huge benefit in terms of anti-tumour activity inclinical trials.

Example 7: PCR Assay for the Identification of IOVA Viruses or theirDerivatives

In order to identify IOVA strain, the DNA of the virus is isolated bydigesting the cell extract of infected cells with proteinase K, and byusing a QIAamp genomic DNA kit (QIAGEN) following manufacturedinstructions. A unique sequence of IOVA covering the C2L-C1L-N1L-N2Lfragment (FIG. 5 a ; SEQ ID No.1) is amplified by PCR using thefollowing oligos: Forward 5′-ATGTTATCCTGGACATCGTAC-3′ (SEQ ID No. 2) andReverse 5′-TCATGACGTCCTCTGCAATGG-3′ (SEQ ID No. 3). The PCR productusing these two primers is 50 bp larger than the unique SEQ ID No.1; forstability reasons, 50 more bp were included in design of the PCRreaction. The PCR product (SEQ ID No.4) is purified by using a QIAquickPCR purification kit and is digested with BstXI restriction enzyme.

By using this assay, IOVA strain can be clearly identified as itgenerates a specific and unique pattern of two DNA bands (2361 and 923bp) visualized by electrophoresis in 1% agarose, in contrast to all theother Vaccinia strains, which present a single band of 3384 bp. PCR ofMVA genomic DNA generates no PCR product due to the absence of the C2Lsequence (FIG. 5 b ).

REFERENCES

D. Kirn, T. Hermiston, F. McCormick, ONYX-015: clinical data areencouraging. Nature medicine 4, 1341 (December 1998).

C. J. Breitbach et al., Oncolytic vaccinia virus disruptstumour-associated vasculature in humans. Cancer research 73, 1265 (Feb.15, 2013).

S. H. Thorne et al., Targeting localized immune suppression within thetumour through repeat cycles of immune cell-oncolytic virus combinationtherapy. Molecular therapy: the journal of the American Society of GeneTherapy 18, 1698 (September 2010).

C. J. Breitbach et al., Intravenous delivery of a multi-mechanisticcancer-targeted oncolytic poxvirus in humans. Nature 477, 99 (Sep. 1,2011).

J. Heo et al., Randomized dose-finding clinical trial of oncolyticimmunotherapeutic vaccinia JX-594 in liver cancer. Nature medicine 19,329 (March 2013).

H. J. Zeh et al., First-in-man study of western reserve strain oncolyticvaccinia virus: safety, systemic spread, and antitumour activity.Molecular therapy: the journal of the American Society of Gene Therapy23, 202 (January 2015).

A. Volz, G. Sutter, Protective efficacy of Modified Vaccinia virusAnkara in preclinical studies. Vaccine 31, 4235 (Sep. 6, 2013).

H. Meyer, G. Sutter, A. Mayr, Mapping of deletions in the genome of thehighly attenuated vaccinia virus MVA and their influence on virulence.The Journal of general virology 72 (Pt 5), 1031 (May 1991).

R. T. Zhang, S. D. Bines, C. Ruby, H. L. Kaufman, TroVax((R)) vaccinetherapy for renal cell carcinoma. Immunotherapy 4, 27 (January 2012).

I. Marigo, L. Dolcetti, P. Serafini, P. Zanovello, V. Bronte,Tumour-induced tolerance and immune suppression by myeloid derivedsuppressor cells. Immunol Rev 222, 162 (April 2008).

S. J. Gardai et al., Cell-surface calreticulin initiates clearance ofviable or apoptotic cells through trans-activation of LRP on thephagocyte. Cell 123, 321 (Oct. 21, 2005).

G. L. Smith et al., Vaccinia virus immune evasion: mechanisms, virulenceand immunogenicity. The Journal of general virology 94, 2367 (November2013).

Goebel et al. The complete DNA sequence of vaccinia virus. Virology 1990November; 179(1): 247-66, 517-63.

L. Galluzzi, A. Buque, O. Kepp, L. Zitvogel, G. Kroemer, Immunogeniccell death in cancer and infectious disease. Nat Rev Immunol 17, 97(February 2017).

Rojas J J et al., Manipulating TLR signaling increases the anti-tumour Tcell response induced by viral cancer therapies. Cell Rep., 2016 Apr.12; 15(2): 264-73.

The invention claimed is:
 1. A genetically modified, immuno-modulatingVaccinia virus, wherein the virus is identified by the presence of SEQID No.: 1, has a functional active K1L, is replication competent inmammalian cells, and induces calreticulin transiocation to the membraneof an infected HeLa cell.
 2. The Vaccinia virus according to claim 1,wherein the virus has, additionally, a functionally inactivated A56R. 3.The Vaccinia virus according to claim 1, wherein the virus has afunctionally inactivated A26R.
 4. The Vaccinia Virus according to claim1, wherein the virus additionally comprises one or more functionallyinactivated immune-evasion genes selected from the group of open readingframes consisting of B21R, C10L, C9L, C4L, M1L, A51R, A52R, A55R, andB13R/B14R.
 5. The Vaccinia virus according to claim 1, wherein the viruscomprises, additionally, at least one functionally inactivated,partially deleted or fully deleted gene selected from the groupconsisting of J2R, C11R, and F4L.
 6. The Vaccinia virus according toclaim 1, wherein the virus is replication-competent and lytic incell-cycle-activated cells and/or tumour cells.
 7. The Vaccinia virusaccording claim 1, wherein the virus, upon infection, causes theformation of syncytia.
 8. A nucleic acid sequence or fragment thereofencoding the Vaccinia virus according to claim
 1. 9. A viral vectorcomprising the nucleic acid sequence according to claim
 8. 10. TheVaccinia virus according to claim 1, characterised by carrying one ormore insertion sites with at least one insertion of one or moretransgenes.
 11. The recombinant Vaccinia virus according to claim 10,wherein the transgene is selected from the group comprising genesencoding tumour antigens, tumour associated antigens, disease associatedantigens, and pathogen-derived antigens.
 12. A method of treating asubject, the method comprising administering to the subject the Vacciniavirus according to claim
 1. 13. A method of treating cancer in asubject, the method comprising administering to the subject the Vacciniavirus according claim
 1. 14. A pharmaceutical composition comprising theVaccinia virus according to claim 1 and a pharmaceutically acceptablecarrier, diluent, or excipient.